Parathion (0,0-diethyl-O-p-nitrophenyl phosphorothioate) was discovered in 1944 by Schrader (Eto, M., 1974 organophosphorus pesticides: organic and biological chemistry, CRC Press Inc., Cleveland, Ohio). It is an organophosphate pesticide that is extremely toxic to higher organisms because parathion (PAR) is converted by the cytochrome P-450 monooxygenase system to paraoxon which is a potent acetylcholinesterase inhibitor (Taylor, The pharmacological basis of therapeutics, 6th ed. pp. 100-119 (1980)).
Organophosphate pesticides applied to agricultural environments at recommended rates are not extremely persistent. However, when higher concentrations of PAR are applied to soil samples, persistence increases, and PAR can be detected in some soils after 16 years (Wolfe et al., Bull. Environm. Contam. Toxicol. 10:1-9 (1973)). It has been estimated that the remaining pesticide residue in pesticide containers is approximately 4.5.times.10.sup.5 kilograms per year (Munnecke, Process Biochem. 13:16-19 (1978)). The major source of pesticide discharges occurs at pesticide production and formulation plants, which release more than 4.times.10.sup.5 kilograms of pesticides each year (Munnecke, Supra (1978)). Therefore, hazards exist with respect to undesirable discharges such as pesticide containers, production plants, or accidental spills (Munnecke, Microbial degradation of xenobiotics and recalcitrant compounds, Academic Press Inc., London, pp. 251-270 (1981)). In these instances, clean-up procedures are required. Enzymes which can decrease the toxicity of the pesticide molecule by a simple hydrolysis reaction could possibly be used for the detoxification of residual pesticides at these industrial point sources.
1. Microbial Degradation of Organophosphates and Possible Industrial Applications PA0 2. Plasmids Involved in Organophosphate Degradation PA0 3. Construction of Recombinant Plasmids Containing the Parathion Hydrolase Gene
Over millions of years, microorganisms have evolved the ability to utilize many compounds as sole sources of carbon and energy (Gibson et al., Microbial degradation of organic compounds, Marcel Dekker, Inc. New York (1984)). The importance of the studies of microbial metabolism is exemplified by the virtue of microorganisms as the major means by which many chemical pollutants are eliminated from a variety of ecosystems.
The vast majority of the 500 or so active pesticidal chemicals can be biologically degraded by fungal or bacterial microorganisms. General reviews discussing microbial metabolism of pesticides have been edited by Bollag, Advan. in App. Micro. 18:75-130 (1974), and Laveglia and Dahm, Annu. Rev. Entomol. 22:483-513 (1977).
Munnecke and Hsieh, Appl. Environ. Microbiol. 31:63-69 (1976) have studied the degradation of parathion by a mixed microbial culture and found the major metabolites to be p-nitrophenol and diethylthiophosphoric acid (FIG. 1). Hydrolysis of parathion reduces its toxicity by nearly 120-fold and releases water-soluble metabolites that are more available to further microbial degradation. p-nitrophenol can be further utilized as a source of carbon and energy by other microorganisms (Munnecke and Hsieh, supra: Simpson, Biochem, J. 55:xxiv (1953); and Spain et al., Biochem. Biophys. Res. Commun. 88:634-641 (1979)). Another study has shown that diethylthiophosphoric acid is utilized as the sole phosphorus source by some soil microorganisms (Cook et al., Appl. Environ. Microbiol. 36:668-672 (1978)).
A cell-free enzyme preparation obtained by Munnecke from a mixed bacterial culture hydrolyzed the organophosphate insecticides ethyl parathion, methyl parathion, paraoxon, diazinon, dursban, EPN, cyanophos, fenitrothion, and triazophos at rates significantly higher than chemical hydrolysis with sodium hydroxide (Munnecke, Appl. Environ. Microbiol. 32:7-13 (1976)). The enzyme activity for pesticide hydrolysis was stable at temperatures up to 45.degree.-50.degree. C. and had a temperature and pH optimum for expression of enzymatic activity of 35.degree. C. and pH 8.5-9.0, respectively (Munnecke, supra (1976)).
Parathion is enzymatically hydrolyzed to diethylthiophosphoric acid (I) and p-nitrophenol (II) by parathion hydrolase for P. diminuta MG (pCMS1) (FIG. 1).
The enzymatic removal of PAR from pesticide containers has been demonstrated (Munnecke, Agric. Food Chem. 28:105-111 (1980)). It was observed that within 16 hours, 94% of the residual PAR present in the drums as a 48% emulsifiable concentrate was hydrolyzed after parathion hydrolase was added.
The feasibility of using enzyme preparations to clean up soil spills has been demonstrated in both laboratory and field conditions. Work by Batik and Munnecke, Bull. Environm. Contam. Toxicol. 29:235-239 (1982) has shown that parathion hydrolase can hydrolyze diazinon in soil. More than 98% of 1% diazinon in the soil was removed within 24 hours when sufficient amounts of buffer and enzyme were added to the contaminated soil. This concentration of pesticide would normally remain in untreated soil for many years (Wolfe et al., supra). In greenhouse studies, soil samples were treated with different concentrations of diazinon (Honeycutt et al., ACS Symposium Series, No. 259, Treatment & Disposal of Pesticide Wastes, pp. 343-352 (1984)). Parathion hydrolase was added to the soil samples to determine the efficacy of the enzyme to rapidly degrade diazinon during a spill situation. The half-life of diazinon in the 500 ppm treatment without enzyme present was 9.4 days, while the half-life of diazinon in the 500 ppm treatment with enzyme present was one hour. These studies indicate that parathion hydrolase can be used effectively to rapidly reduce large concentrations of diazinon in soil.
Investigations using a mixed microbial culture that converts PAR to PNP led to the isolation of Pseudomonas diminuta strain MG which contains high levels of parathion hydrolase activity (Serdar et al., Appl. Environ. Microbiol. 44:246-249 (1982)). In all cases, parathion hydrolase activity was produced in the absence of added PAR. On the basis of these results, it appears that parathion hydrolase was produced constitutively by this organism. P. diminuta MG and a Flavobacterium sp. (ATCC 27551), isolated by Sethunathan and Yoshida, Can. J. Microbiol. 19:873-875 (1973), are the most extensively studied organisms that degrade organophosphates.
Plasmids are extrachromosomal genetic elements that code primarily for nonessential functions yet enable certain organisms to survive in unique ecological niches. Many unusual metabolic activities of Pseudomonas species are plasmid-encoded functions (Haas, Experentia, 39:1199-1213 (1983)).
To determine whether parathion hydrolase activity was controlled by a plasmid, attempts have been made to correlate loss of PAR hydrolase activity with plasmid removal. Expression of enzymatic activity was lost at a frequency of approximately 12% after treatment with mitomycin C (Serdar, supra (1982)). Hydrolase-negative derivatives were lacking a plasmid present in the wild-type organism. These cured colonies differed phenotypically from the parent strain only in inability to hydrolyze PAR. These results suggested that PAR hydrolase activity may be mediated by plasmid DNA, although induction of deletion mutations could not have been excluded.
This plasmid designated as pCMS1 was determined to be 66 kilobase (kb) in size by using electron microscopy (Serdar, supra (1982)). Recent studies using restriction mapping have confirmed the molecular size estimate (Mulbry et al., Appl. Environ. Microbiol. 51:926-930 (1986)). This is the first example of plasmid involvement in the degradation of an organophosphorus compound.
More recently it was shown that the Flavobacterium sp. ATCC 27551 contains a 39 kb plasmid, pPDL2, that is involved in the degradation of organophosphates (Mulbry et al., supra (1986); and Mulbry et al., Plasmid 18:173-177 (1987)). The gene encoding parathion hydrolase (termed opd, organophosphate-degrading gene) from pCMS1 was shown to be homologous to that located in pPDL2, as determined by DNA--DNA hybridization and restriction mapping. Further DNA hybridization studies have revealed that these plasmids share homology only within an approximately 5.1 kb region of DNA (Mulbry et al., supra (1987)).
Two cloning stategies were employed (Serdar and Gibson, Bio/Technology 3:567-571(1985)). One method was the use of different restriction endonuclease sites of pBR322 (Bolivar et al., Gene. 2:95-113(1977)). for `shot-gun` cloning with subsequent studies for parathion hydrolase expression in E. coli. The other procedure utilized the EcoRI site of the broad host range vector, pKT230 (Bagdasarian et al., Gene. 16:237-247(1981)); recombinant plasmids generated using pKT230 can be mobilized into Pseudomonas strains using a helper plasmid, pRK2013 (Figurski et al., Proc. Natl. Acad. Sci. USA. 76:1648-1652(1979)).
Following the cloning experiment using pKT230 as the vector, two transformants that hydrolyzed PAR were isolated, and each was shown to contain a hybrid plasmid with an identical 6.0 kilobase EcoRI insert (Serdar, supra(1985)). This plasmid was designated as pCMS29 as shown in FIG. 2.
FIG. 2 represents the physical maps of the recombinant plasmids carrying the parathion hydrolase gene of P. diminuta MG (pCMS1). The thick lines represent the adjacent portions of vector DNA; the thin lines represent the inserted pCMS1 DNA fragments. Restriction endonucleases used were: B, BamHI; E, EcoRI; H, HindIII; P, PstI; S, SalI; T, TaqI; X, XhoI.
Alternative cloning procedures utilized the BamHI, HindIII and SalI restriction sites of the vector pBR322. DNA fragments produced from pCMS1 by these restriction enzymes were ligated into similarly treated pBR322 and were used to transform E. coli HB101. A BamHI-generated clone which hydrolyzed PAR was shown to contain a hybrid plasmid designated as pCMS5 (FIG. 2). Analysis by restriction endonuclease digestion revealed that this plasmid contained a 1.5 kb insert. In contrast, parathion hydrolase activity was not detected in any of the transformants containing hybrid plasmids constructed with SalI.
In order to increase the synthesis of parathion hydrolase in E. coli and to construct a 1.5 kb fragment suitable for inserting into the EcoRI site of the plasmid pKT230, the 1.5 kb BamHI fragment located in pCMS29 was subcloned into the high expression vector pUC7 (Vieira et al., Gene. 19:259-268(1982)). This resulting strain contained a plasmid, designated as pCMS40, with a 1.5 kb insert which was flanked by the EcoRI sites of pUC7. Restriction maps of the 6.0 kb EcoRI fragment in pCMS29, the 1.5 kb BamHI fragment in pCMS5, and the subcloned 1.5 kb BamHI fragment in pCMS40 are shown in FIG. 2.
Plasmids suitable for studying the expression of the parathion hydrolase gene in Pseudomonas strains were constructed by EcoRI cleavage of pCMS40 and subsequent ligation into EcoRI cleaved pKT230.
TABLE I ______________________________________ Parathion hydrolase activity* (Prior Art) Activity ______________________________________ Strain P. diminuta MG ND P. diminuta MG (pCMS1) 2.1 P. diminuta MG (pCMS29) 4.8 P. diminuta MG (pCMS55) 9.2 Pseudomonas strain 24 ND Pseudomonas strain 24 (pCMS29) 0.03 Pseudomonas strain 24 (pCMS55) 0.07 E. coli strains HB101 (pBR322) ND BHB2600 (pKT230) ND JM105 (pUC7) 0.002 BHB2600 (pCMS29) 0.002 HB101 (pCMS5) 0.03 JM105 (pCMS40) 0.19 BHB2600 (pCMS55) 0.004 BHB2600 (pCMS60) ND ______________________________________ *Activity has been converted from nanomoles to micromoles (.mu.mol) of parathion hydrolyzed per minute per milligram of protein. ND, not detected.
E. coli BHB2600 cells transformed with plasmids designated pCMS55 and pCMS58 showed enzymatic activity, and each plasmid contained the 1.5 kb BamHI insert. The highest parathion hydrolase activity was associated with pCMS55 which contained two 1.5 kb inserts in the same orientation relative to the vector as shown in FIG. 3. Further screening led to the isolation of an additional strain that contained a plasmid (pCMS60) with the same 1.5 kb insert in the opposite orientation. However, this strain showed no parathion hydrolase activity. FIG. 3 represents the physical maps of pKT230 derivatives containing the parathion hydrolase gene in different orientations. The heavy lines correspond to the pKT230 DNA; the thin lines show the inserted pCMS1 DNA. Restriction endonucleases used were: B, BamHI; E, EcoRI, P, PstI; S, SalI; X, XhoI.
Of particular interest to the background of the present invention is McDaniel et al., J. Bacteriol., 170:2306, 1988, which published a DNA sequence of a 1.32 kb PstI fragment from P. diminuta MG plasmid, pCMS1. McDaniel et al. observed the total number of base pairs (bp) to be 1322. According to their analysis the stop codon (TGA) was located at the unique Ddel site within the parathion hydrolase gene at 1038 bp. The open reading frame was determined to be 975 bp. McDaniel et al. constructed several subclones: two clones extended the 3'-end to approximately 675 bp and 840 bp. These strains, containing either clone, had no enzymatic activity. Another construct was deleted at the 5'-end SalI site (353 bp) which also did not contain activity.
McDaniel et al. concluded that the parathion hydrolase enzyme was not soluble and was associated with the particulate fraction of lysed P. diminuta cells. They suggested that the enzyme was released from the particulate fraction upon treatment with detergents such as Triton X-100 or Tween 20. From their evaluation of elution fractions from Sephadex G-200 column chromatography, they concluded that the molecular weight of the enzyme was 60-65 kilodaltons (kd). From their molecular weight determinations, McDaniel et al. suggested that the enzyme exists in its active form as a dimer. The McDaniel et al. article provides no evidence that the enzyme produced is processed parathion hydrolase.
To date, a correct gene encoding sequence for unprocessed parathion hydrolase has not been described. In particular, no gene encoding sequence for processed parathion hydrolase has been described or suggested.